Lysosomes carry out degradative metabolism critical to many endocytic, phagocytic, and autophagic processes. Mannose 6-phosphate receptors (CI-MPR and CD-MPR) play a vital role in the biogenesis of lysosomes by delivering ~60 different newly synthesized hydrolytic enzymes with mannose 6- phosphate on their N-glycans to lysosomes. CI-MPR is the primary receptor responsible for this trafficking and the ability of CI-MPR to internalize these enzymes is capitalized upon in enzyme replacement therapy for several lysosomal storage diseases. Unlike CD-MPR, CI-MPR binds a diverse set of extracellular ligands that mediate CI-MPR's ability to function as a tumor suppressor and to regulate cell growth and differentiation. CI-MPR interacts with three components of the plasminogen activation system, plasminogen, urokinase-type plasminogen activator receptor (uPAR), and tissue-type PA (tPA). Previous studies showed differentiation of fibroblasts to myofibroblasts, a key process during wound healing that becomes aberrant in fibrosis, requires the cleavage of uPAR and decreased levels of surface-bound uPAR. Recently we showed that CI-MPR is required for the differentiation of human corneal fibroblasts to myofibroblasts. However, limited information is available concerning the molecular basis for CI-MPR's interaction with plasminogen and uPAR and how CI- MPR impacts cellular differentiation. In this proposal, we expand upon our preliminary data that reveal the first structural view of the entire extracellular region of CI-MPR by high-resolution electron microscopy (EM), and our identification of a new ligand (tPA) for CI-MPR that enhances plasminogen activation upon binding CI-MPR. The overall structure of CI-MPR's 2300-residue extracellular region comprised of 15 domains will be determined using an integrated approach combining single particle EM, X-ray crystallography and NMR spectroscopy (Aim 1). The first structure of a complex between CI-MPR and a lysosomal enzyme will be elucidated using EM and crystallographic approaches (Aim 1). The mechanism of acidic pH-dependent ligand dissociation, which is essential to the functioning of the MPRs and other endocytic receptors, will be probed using mutagenesis studies and NMR techniques (Aim 1). The role of tPA and uPA in cellular differentiation will be investigated (Aim 2). The structure of a complex between CI-MPR and uPAR, and CI-MPR and plasminogen will be solved by crystallographic and NMR methods (Aim 2). Cell-based assays and quantitative binding studies will be done to evaluate: 1) how the different ligands of CI-MPR impact CI-MPR's function in ligand binding and internalization (Aim 1), and 2) whether CI-MPR regulates uPAR's half-life and function (Aim 2). These studies will provide insight for the design of improved therapeutics for the treatment of lysosomal storage diseases, and novel inhibitors of the plasminogen activation system.